Fluorescence study of domain structure and lipid interaction of human apolipoproteins E3 and E4

Abstract

Human apolipoprotein E (apoE) isoforms exhibit different conformational stabilities and lipid-binding properties that give rise to altered cholesterol metabolism among the isoforms. Using Trp-substituted mutations and site-directed fluorescence labeling, we made a comprehensive comparison of the conformational organization of the N- and C-terminal domains and lipid interactions between the apoE3 and apoE4 isoforms. Trp fluorescence measurements for selectively Trp-substituted variants of apoE isoforms demonstrated that apoE4 adopts less stable conformations in both the N- and C-terminal domains compared to apoE3. Consistent with this, the conformational reorganization of the N-terminal helix bundle occurs at lower guanidine hydrochloride concentration in apoE4 than in apoE3 as monitored by fluorescence resonance energy transfer (FRET) from Trp residues to acrylodan attached at the N-terminal helix. Upon binding of apoE3 and apoE4 variants to egg phosphatidylcholine small unilamellar vesicles, similar changes in Trp fluorescence or FRET efficiency were observed for the isoforms, indicating that opening of the N-terminal helix bundle occurs similarly in apoE3 and apoE4. Introduction of mutations into the C-terminal domain of the apoE isoforms to prevent self-association and maintain the monomeric state resulted in great increase in the rate of binding of the C-terminal helices to a lipid surface. Overall, our results demonstrate that the different conformational organizations of the N- and C-terminal domains have a minor effect on the steady-state lipid-binding behavior of apoE3 and apoE4: rather, self-association property is a critical determinant in the kinetics of lipid binding through the C-terminal helices of apoE isoforms.

Helical structure, stability, and dynamics in human apolipoprotein E3 and E4 by hydrogen exchange and mass spectrometry

“…3 and 4 are consistent with ΔG of helix stabilization in the range 4 kcal/mol to 5 kcal/mol. This same range holds for the N-domain helix bundle in the monomeric and tetrameric states of apoE3 and apoE4 at 5°C and 25°C, and is similar to values obtained by urea denaturation measurements with both isoforms (4 kcal/mol to 7 kcal/mol) (15,43). This stabilization ΔG is consistent with the existence of mutually stabilizing helix−helix interactions in the helix bundle, (31) because individual unsupported α-helices are not stable.…”

“…The above alterations in conformation must underlie the reduced ability of apoE4 to form tetramers relative to apoE3 (15,28). Although the structure of dimeric and tetrameric apoE is unknown, the concept that the reduced C-domain helix length in apoE4 (Fig.…”

“…As summarized in the introductory paragraphs, the physiological functions of the apoE molecule involve various binding events that are mediated by amphipathic α-helices located in the N-and C-terminal domains. After dissociation of apoE tetramers [such as occur in plasma HDL-LpE (35)] to the monomeric state (15,47), the C-terminal helical domain initiates hydrophobic interactions such as binding to lipid and lipoprotein surfaces (7,44), and to amyloid-β (13,48). The present HX results prove that the amphipathic α-helix organization in the C-terminal domains of tetrameric WT apoE3 and apoE4 is different and suggest the following explanation for the molecular basis of the enhanced binding of apoE4 in such situations.…”

“…This parallelism occurs because of altered domain−domain interactions in the apoE3 and E4 isoforms, variously explained by either altered salt-bridge formation (25) or allosteric effects (22,23,26). The bulky charged arginine residue in apoE4 acts directly to destabilize the N-terminal helix bundle and, indirectly, the C-terminal domain (15,27).…”

“…A highresolution structure of the wild-type (WT) apoE molecule is unavailable because both apoE3 and E4 self-associate through their C-terminal domains to form a tetramer, which has inhibited study by X-ray crystallography and NMR. It has been possible to obtain the structures of the N-and C-terminal domains using a monomeric variant with five mutations (F257A/W264R/V269A/L279Q/V287E) in the C-terminal domain that interfere with tetramer formation while maintaining similar lipid-binding activity (14,15). NMR study of the monomeric variant of human apoE3 (16) and crystallography of the isolated N-terminal domain (1,17) show that residues 23-167 form an N-terminal antiparallel four-helix bundle.…”